This R01 application entitled Lead Optimization of Inhibitors of the Thioesterase Domain of Fatty Acid Synthase is in response to PAR-12-060 Solicitation of Validated Hits for the Discovery of in vivo Chemical Probes. Metabolic re-wiring is now recognized as one of the hallmarks of cancer and can be observed in many sub-networks of central carbon metabolism. The lipogenic sub-network is often up-regulated in solid tumors, and increased expression and activity of fatty acid synthase (FASN) is required for the survival and proliferation of many tumor cells, including prostate, breast, colon, ovaries, and liver. Importantly for this proposal, a clear role for FASN has recently been established in malignant melanoma, a life-threatening form of skin cancer. Thus, inhibition of FASN is a promising approach for the treatment of multiple very serious forms of cancer, and especially melanoma. We recently screened 360K compounds against the thioesterase (TE) domain of FASN through the MLPCN program and identified several small molecule hits that proved to be tractable. In particular, one scaffold yielded a family of analogues with promising in vivo properties. The most advanced compound from this series is a potent and highly selective inhibitor of FASN-TE in vitro, blocks fatty acid biosynthesis in whole cells, halts tumor cell proliferation, is non-toxic in normal cells, and shows promising drug levels in mice following a single systemic dose (10 mg/kg i.p.). However, high protein binding, poor aqueous solubility and low microsomal stability suggest that the pharmacokinetic properties must be optimized to provide compounds suitable for in vivo proof-of-concept experiments. These compounds are ready for full- scale chemistry optimization to provide lead compounds ready for in vivo proof-of-concept studies. Therefore our Specific Aims are: 1. Design and synthesize optimized FASN-TE inhibitors that are orally active in vivo. 2. Assess potency and selectivity of FASN-TE inhibitors in relevant in vitro and cellular assays. 3. Evaluate FASN-TE inhibitors using in vitro ADME/T and in vivo pharmacokinetic (PK) assays. 4. Determine efficacy of lead FASN-TE inhibitor probes in relevant mouse tumorigenic (xenograft) models. The FASN-TE inhibitors generated will provide powerful tools for testing the hypothesis that inhibition of FASN is an effective method for killing tumor cells, while laying a foundation for future development of a novel class of medications for the treatment of cancer.